Autoradiographic Localization of Sigma Receptor ... - Semantic Scholar

The Journal of Neuroscience June 1966, t?(6): 1757-1770

Autoradiographic Localization of Sigma Receptor Binding Sites in Guinea Pig and Rat Central Nervous System with ( +)3H-3-(3-Hydroxyphenyl)4V-( 1 -propyl)piperidine Andrew L. Gundlach,’ Brian L. Largent, and Solomon H. Snyder Departments of Neuroscience, Pharmacology and Experimental Therapeutics, Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

( +)3H-3-PPP [( +)3H-3-(3-Hydroxyphenyl)-N-(l-propyl)-piperidine] binds with high affinity to brain membranes with a pharmacological profile consistent with that of sigma receptors. The distribution of (+)3H-3-PPP binding sites in brain and spinal cord of both guinea pig and rat has been determined by in vitro autoradiography with binding densities quantitated by computer-assisted densitometry. ( +)3H-3-PPP binding to slidemounted brain sections is saturable and displays high affinity and a pharmacological specificity very similar to sites labeled in homogenates. (+)‘H-3-PPP binding sites are heterogeneously distributed. Highest concentrations of binding sites occur in spinal cord, particularly the ventral horn and dorsal root ganglia; the pons-medulla, associated with the cranial nerve and pontine nuclei and throughout the brain stem reticular formation; the cerebellum, over the Purkinje cell layer; the midbrain, particularly the central gray and red nucleus; and hippocampus, over the pyramidal cell layer. Lowest levels are seen in the basal ganglia and parts of the thalamus, while all other areas, including hypothalamus and cerebral cortex, exhibit moderate grain densities. Quinolinic acid-induced lesions of the hippocampus indicate that ( +)3H-3-PPP labels hippocampal pyramidal cells and granule cells in the dentate gyrus. Intrastriatal injection of ibotenic acid dramatically reduces ( +)3H-3-PPP binding in this area, while injection of 6-hydroxydopamine produces a relatively slight decrease.The distribution of (+YH-3-PPP binding sites does not correlate with the receptor distribution of any recognized neurotransmitter or neuropeptide, including dopamine. However, there is a notable similarity between the distribution of (+)3H-3-PPP sites and high-affinity binding sites for psychotomimetic opioids, such as the benzomorphan (+)SKF 10,047.

have been noted between the behavioral effects in animals and the psychotomimetic effects in man caused by such drugs (Haertzen, 1970; Keats and Telford, 1964). Many pharmacological effects of drugs such as SKF 10,047 and cyclazocine are not blocked by opioid antagonists, e.g., naloxone or naltrexone (Iwamoto, 198 1; Vaupel, 1983; Young and Khazan, 1984), suggesting that these drugs interact at sites very different from more classical opiate receptors. Furthermore, the ability of benzomorphans to produce “sigma” or psychotomimetic-like behavioral effects generally resides more in the (+) than the (-) isomer (Brady et al., 1982; Katz et al., 1985; Slifer and Balster, 1983), a stereoselectivity opposite to that found at mu, kappa, or delta opiate receptors. Binding sites in brain membranes with a drug specificity consistent with putative sigma receptors were first identified with (f)3H-SKF 10,047 in the presence of unlabeled etorphine (Su, 198 1, 1982). Sigma receptor binding sites have also been labeled with (+)3H-SKF 10,047 (Gundlach et al., 1985; Largent et al., in press; Martin et al., 1984; Tam, 1983, 1985; Tam and Cook, 1984), (+) and (+)3H-ethylketocyclazocine (Tam, 1983, 1985) and )H-haloperidol (Largent et al., 1984; Tam and Cook, 1984). Binding of (+) and (-)3H-SKF 10,047 to mouse brain membranes reveals that the isomers differ in drug specificity and regional distribution (Martin et al., 1984). Similarities in biochemical and behavioral effects of sigma opioids and phencyclidine (PCP) have suggested actions through a common receptor designated, sigma opioid/PCP receptor (Shannon, 1983; Zukin and Zukin, 198 1). However, the regional distribution and pharmacology of (+)3H-SKF 10,047-labeled sigma receptor binding sites differ from ‘H-PCP-labeled sites (Gundlach et al.,

The existence of three types of opioid receptors-mu, kappa, and sigma-was postulated to explain the effects of different opioids in chronic spinal dog preparations (Martin et al., 1976). Sigma receptors, named for the prototypic agonist SKF 10,047 (N-allylnormetazocine), were suggested to mediate the psychotomimetic-like effects of SKF 10,047 in the dog. Similarities

Table 1. Comparisonof drug potenciesin inhibiting (+)3H-3-PPP binding to brain sectionsand homogenates

Drug

Brain section

Brain homogenate”

Received Oct. 14, 1985; accepted Nov. 26, 1985. This work was supported by USPHS Grants DA-00266 and NS-16375, and Research Scientist Award DA-00074 to S.H.S.; National Institutes of Health Training Grant GM-07626 to B.L.L.; and a grant from the M&night Foundation. A.L.G. is the recipient of a National Health and Medical Research Council (Australia) C. J. Martin Fellowship. We wish to thank Dawn C. Dodson and Ida M. Anderson for secretarial assistance and Paul F. Worley and Donald C. Manning for help with lesion studies and for providing spinal cord sections, respectively. Correspondence should be addressed to Solomon H. Snyder, Departments of Neuroscience, Pharmacology and Experimental Therapeutics, Psychiatry and Behavioral Sciences,The John Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. ’ Present address: Department of Pharmacology, University of Sydney, Sydney, N.S.W. 2006, Australia. Copyright 0 1986 Society for Neuroscience 0270-6474/86/061757-14$02.00/O

Haloperidol (-)Butaclamol (+)Butaclamol Pentazocine (+)Cyclazocine (-)Cyclazocine

5.0 x 4.2 x 2.0 x 4.0 x 1.3 x 1.5 x

3.0 x 1.6 x 2.0 x 2.5 x 1.2 x 4.5 x

1757

1% h)

10-g 10-1 10-z 10-a 10-7 10-e

10-Y lo-’ 10-G 10-a 10-7 10-7

Slide-mounted sagittal sections ( 12 pm) of guinea pig brain were incubated with 4-5 nM (+)3H-3-PPP in the presence of Seven (triplicate) or 10 (duplicate) concentrations of drug, as described in Materials and Methods. IC,, values were calculated from the data using the EBDA computer program (McPherson, 1983). Values are the average of two determinations. EValues from Largent et al. (1984).

1758

Gundlach et al.

Vol. 6, No. 6, Jun. 1986

Fzgure 1. Autoradiograms detailingthe anatomicaldistributionof (+)‘H-3-PPPbindingin guineapigbrain(A-L). Representative brainsections

arepresented in a rostral-caudalprogression. Increased whiteness in the photographs, printeddirectly from the Ultrofilm, corresponds to higher levelsof (+PH-3-PPPbinding.As detailedin MaterialsandMethods,8 pm sections wereincubatedwith 5 nM(+PH-3-PPPfor 45 min at room temperature, washed,dried,andapposed to Ultrofilm for 4 weeksat 4°C.Nonspecific bindingwasmeasured in the presence of 1 PM haloperidol andislessthanlabelingseenoverwhitematterin theseplates.Structureswereidentifiedby reference to the atlases of Luparello(1967)andPaxinos n.; AR, anteriorpretectalarea;CAl-3, fieldsCA-l, CA-2, CA-3 of Ammon’s andWatson(1982).A& Accumbens nucleus(n.); Amb, ambiguus Cb, cerebellum; cc,corpuscallosum;CG,centralgray; Cg,cingulatecortex;CP, caudateputamen;DDb, dorsaldiagonalband horn,hippocampus; of Broca;DG, dentategyms;DTg, dorsaltegmentaln.; ECU, externalcuneaten.; Ent, entorhinalcortex; Fuc, facial n.; Fr, frontal cortex; Gn, 1985; Largent et al., in press;Martin et al., 1984; Tam, 1983, 1985; Zukin and Zukin, 1979). 3-(3-Hydroxyphenyl)-N-( 1-propyl)piperidine (3-PPP) and its enantiomersproduce behavioral and biochemical effectsin animals that are consistentwith agonistactions at dopamine autoreceptors.Theseincludein vivo effectssimilarto the dopamine agonistapomorphine,e.g.,decreasednigral dopaminecell firing, reduction in the synthesisrate of dopamine in reserpine- or gammabutyrolactone-treatedrats, and decreasedlocomotor ac-

tivity (Arm et al., 1983;Hjorth et al., 1981, 1983, 1985). However, in vitro, the lack of potency of 3-PPP in experimental systemsthought to measuredopamine autoreceptor activity, such as inhibiting 3H-dopamine release from striatal slices (Markstein and Lahaye, 1983; Mulder et al., 1985; Sminia and Mulder, 1983) and decreasingtyrosine hydroxylase activity in synaptosomes(Haubrich and Pflueger, 198l), is inconsistent with the in vivo findings and callsinto questionthe mechanism underlying 3-PPP’sin vivo effects.

The Journal of Neuroscience

Sigma Receptor Distribution in CNS

granule cell layer, cerebellum; GP, globus pallidus; Hb, habenula; Hpg, hypoglossal n.; ZC, inferior colliculus; ZN, zona incerta; LG, lateral geniculate n.; Lh4, lateral mamillary n.; LS, lateral septal n.; LtC, lateral cervical n.; LtR, lateral reticular n.; MC, medial geniculate n.; MO, molecular layer, cerebellum; MPo, median preoptic n.; MS, medial septal n.; MT, motor trigeminal n.; oc, optic chiasm; PCg, posterior cingulate cortex; Pk, Purkinje cell layer, cerebellum; Pn, pontine n.; PnR, pontine reticular n.; PO, preoptic area; PO, primary olfactory cortex; PrH, prepositus hypoglossal n.; Py, pyramidal cell layer, primary olfactory cortex; Rn, red n.; Rt, reticular n.; Sb, subiculum; SC, superior colliculus; SN, substantia nigra; SO, supraoptic hypothalamic n.; St, striate cortex; TRt, reticular thalamic n.; Ve, vestibular n.; VDb, ventral diagonal band of Broca; VP, ventroposterior thalamic area. Further details are given in the text. Calibration bar, 3 mm.

Recently, we identified specific binding sites for (+)3H-3-PPP in brain (Largent et al., 1984). These apparently non-dopami-

nergic binding sites have a pharmacological profile and regional distribution essentially the same as putative sigma receptor sites labeled with ( +)3H-SKP 10,047 (Gundlach et al., 1985; Largent et al., in press; Martin et al., 1984; Tam, 1983, 1985). Preliminary autoradiographic

studies revealed

an association

of (+)‘H-

3-PPP binding sites with limbic and motor areas (Largent et al., 1984). The present study describes in detail the distribution of

these binding sites in guinea pig and rat CNS as determined by in vitro autoradiography in combination with selective neuronal lesions. Materials

and Methods

Tissuepreparation Male Hartley guinea pigs, 250-350 gm, or male Sprague-Dawley rats, 200-250 gm (Hilltop, Scottsdale, PA), were used in all studies. Animals

1760

Gundlach et al.

Table 2. Regional distribution

of (+pH-3-PPP

binding sites in guinea pig CNS Binding (fmol/mg

Region Cortex

protein)

Region

Binding (fmobmg

Midbrain

Frontal Frontoparietal Pyriform, pyramidal Entorhinal Cingulate Anterior Posterior

cell layer

85 94 125 84

k 8 + 6 f 28 3~ 6

88 * 4 190 f 6

Basal ganglia Caudate putamen Accumbens n.U Globus pallidus Septal area Lateral septal n. Medial septal n. Diagonal band of Broca Vertical limb Horizontal limb Septohypothalamic n. Ventral pallidum Septofimbrial n. Bed nucleus stria terminalis Hippocampal

Vol. 6, No. 6, Jun. 1986

73 f 4 70 zk 2 77 zk 3

Amy&h Central n. Medial n. Anterior cortical n.

221 143 217 138 116 195

* * -c k + f

22 20 40 14 15 48

144 46 126 144

f f + k

50 10 14 6

107 f 16 108 f 14 98 + 10

Thalamus Habenular n. Medial geniculate n. Lateral geniculate n. Reticular n. Anteromedial n. Ventral thalamic n. Zona incerta

184 93 100 206 123 76 155

zk 9 f 4 + 5 + 7 + 3 k 7 f 4

182 150 161 221 221 107 160 134

f 40 + 24 + 11 rf: 52 f 45 + 10 + 15 z!z 15

Hypothalamus Anterior hypothalamic area Paraventricular n. Lateral preoptic area Medial preoptic area Supraoptic n. Mamillaty n. Lateral mamillary n. Medial forebrain bundle

159 118 291 172 160 121 182 207 325 268 251 178

zk 18 k 6 + 10 zk 6 + 6 f 50 + IO + 10 f 42 f 44 z!z57 + 22

392 523 802 275 305 240 301 784

k i + + f k + f

245 373 162 376 241 262 242 251 291 201

f 15 + 43 f 56 f 26 + 36 + 28 + 7 f 23 f IO *9

520 126 250 319 171

f + f * *

18 30 40 10 41

330 104 191 134

k f * f

18 7 17 13

Cranial nerve nuclei 130 _t 11 223 + 15

formation

Stratum pyramidale Stratum radiatum Dentate gyrus, granular layer Subiculum

Superior colliculus Inferior colliculus Red n. Substantia nigra Ventral tegmental area of Tsai Interpeduncular n. Lateral tegmental n. Dorsal tegmental n. Anterior pretectal area Central gray Dorsal raphe n. Deep mesencephalic n. Oculomotor n. (3) Trigeminal motor n. (5) Facial n. (7) Medial vestibular n. (8) Superior vestibular n. (8) Dorsal motor and solitary n. (10) Ambiguus n. (10) Hypoglossal n. ( 12) Pons Pontine central gray Pontine reticular n. Pontine area Reticulotegmental n. Locus coeruleus Cuneiform n. Gigantoccllular reticular n. Paragigantocellular reticular n. Ventral parabrachial n. Dorsal parabrachial n.

55 36 45 15 7 18 40 43

Medulla Prepositus hypoglossal n. Deep cochlear n. Ventral cochlear n. External cuneate n. Parvocellular reticular n. Cerebellum Purkinje layer Molecular layer Granular layer Interpositus cerebellar n. Spinal cord Ventral bomb CeNical Thoracic

Lumbar

965 + 95 895 * 58 618 + 88

protein)

The Journal

of Neuroscience

Sigma

Receptor

Distribution

in CNS

1761

Table 2. Continued Binding (fmol/mg

Region Dorsal home Cervical Thoracic Lumbar Spinal root ganglion Dorsal Ventral

protein)

Binding (fmoVmg

Region

protein)

White matter tracts 164 + 8 201 k 45 100 ? 25 1172 + 190 1198 ? 138

Corpus callosum Anterior commissure Fomix Optic tract Spinal white matter

30 40 32 32 38

f 12 + 3 k 6 rfr 8 f 4

Tissue sections (8 wm) were labeled, as described in Materials and Methods, with 5 no (+)‘H-3-PPP and apposed to LKB Ultrofilm for 4 weeks to generate autoradiograms. Nonspecific binding was defined as that in the presence of 1 pi haloperidol or 20 PM (+)cyclazocine, which gave equivalent low levels in all serial sections examined. Autoradiograms were analyzed using a computerized microdensitometer (Kuhar et al., 1984), and optical densities were converted to fmol/mg protein by reference to lH-microscale standards (Geary and Wooten, 1983; Unnerstall et al., 1982). Multiple readings were taken for each brain strncture on any individual autoradiogram and two-four sections at the same brain level from at least two animals were examined. Brain structures were identified by reference to the atlases of Luparello (1967) and Paxinos and Watson (1982). 0 n. = nucleus or nuclei. hValues represent densities over large cells in ventral horn. j Values represent densities for overall gray matter.

were anesthetized with sodium pentobarbital and perfused via the left cardiac ventricle or aorta with 200 ml 50 mM sodium phosphate, pH 7.5, containing 100 mM NaCl, followed by 200 ml 50 mM sodium phosphate, pH 7.5, containing 0.3 M sucrose. After decapitation, brains and/or spinal cords were removed and mounted in a 1: 1 mixture of bovine brain paste : Tissue-tek (Miles Laboratories, Naperville, IL) in plastic embedding molds and frozen rapidly on dry ice-ethanol. At this stage, tissues were stored at - 20°C. Tissues were mounted on microtome chucks, and 8 or 12 pm sections were cut at - 18°C and thaw-mounted on gelatin/chrome alum-coated slides. Brain sections were stored at -20°C until use.

Autoradiography Tissue sections were warmed to room temperature and washed in 50 mM Tris-HCI buffer, pH 8.0 (incubation buffer), for a 15 min preincubation period at 22-25°C. In routine experiments, slides were drained and transferred to the incubation mixture in 50 ml coplin jars. Brain sections were incubated in buffer containing 4-5 nM (+)‘H-3-PPP for 45 min at 22-25°C. Nonspecific binding was determined by inclusion of 1 MUMhaloperidol or 20 PM (+)cyclazocine in the incubation buffer. For drug-inhibition studies, brain sections after preincubation were dried on a slide-warmer set at 30°C and then covered with 300-500 ~1 of incubation buffer containing 4-5 nM (+PH-3-PPP and various concentrations of drug for 45 min at 22-25°C. Following incubation, slides were dipped (l-2 set) and then washed (2 x 4 min) in buffer, rinsed in distilled water, and dried under a stream of cold dry air and then on a slide-warmer (30°C). Brain sections were then either removed with glass fiber filter disks and their radioactivity content estimated by scintillation spectrometry or apposed to tritium-sensitive Ultrofilm (LKB, Gaithersburg, MD) at 4°C for 4-10 weeks to generate autoradiograms.

Data analysis After the appropriate time of exposure, Ultrofilm was developed and fixed using Kodak D-19 developer and Rapid Fixer. Densitomettic analysis of (+PH-3-PPP binding was performed using a computerized microdensitometer (Loats Associates, Westminster, MD, Kuhar et al, 1984) with optical densities converted to fmol/mg protein using a standard scale based on tritium-methacrylate microscales (Amersham, Chicago, IL; Geary and Wooten, 1983) exposed to each film. Data from drug-inhibition experiments were analyzed using the EBDA curve-fitting computer

program

(McPherson,

1983).

Lesion studies Selective neuronal lesions in different brain areas were made using varMale Sprague-Dawley rats (200-250 gm) were anesthetized by intraperitoneal injection of chloral hydrate (400 mg/kg) or sodium pentobarbital(65 mg/kg) and placed in a stereotaxic frame (Kopf ious neurotoxins.

Instruments,

Tujunga,

CA). Injection-site

coordinates

were determined

using the atlas of Paxinos and Watson (1982) and confirmed injection in age-matched controls or by histological examination sectioning

for autoradiographic

by dye during

studies.

Quinolinic, ibotenic, and kainic acids were dissolved in 0.9% NaCl and adjusted to pH 7.4 with 0.1 M NaOH. 6-Hydroxydopamine was dissolved in a 1 mg’ml ascorbic acid solution. Toxins were administered into the desired brain area in a volume of l-2 ~1 with a Hamilton microsyringe over l-2 min. Lesioned animals were sacrificed at the appropriate time following treatment. The extent and selectivity of lesions was determined by histological examination of brain sections with toluidine blue, acetylcholinesterase, and silver stains (Ralis et al., 1973). Quinolinic acid (200 nmol) was used to lesion the hippocampal formation (Schwartz et al., 1983). Ibotenic acid (100 nmol) or 6-hydroxydopamine (100 nmol) was microinjected unilaterally into the corpus striatum to lesion intrinsic neurons or dopaminergic neurons, respectively

(Schwartz et al., 1979; Ungerstedt,

was performed determine

as described and densitometric

the quantitative

197 1). Autoradiography

analysis was used to

regional effects of the lesions on (+PH-3-

PPP binding.

Materials (+)‘H-3-PPP

(107.4 Ci/mmol)

was kindly provided

by New England

Nuclear Corporation (Boston, MA). (+)3-PPP was generously donated by Astra Laboratories (Sodertalje, Sweden). Drugs and their sources

were as follows: haloperidol, McNeil Pharmaceutical (Spring House, PA); (+) and (-)butaclamol, Research Biochemicals International (Wayland, MA); (*)pentazocine and (+), (-)cyclazocine, Sterling Winthrop Research Institute (Rensselaer, NY). Quinolinic acid and 6-hydroxydopamine were purchased from Sigma Chemical Company (St. Louis, MO), and ibotenic acid was obtained from Regis Chemical Company (Morton Grove, IL). Results

General characteristics of (+)‘H-3-PPP binding to brain sections Before attempting autoradiographic localization of (+PH-3-PPP binding sites, it was necessary to establish that binding sites labeled with (+PH-3-PPP in slide-mounted brain sections have similar properties to those in tissue homogenates (Table 1). In brain sections, (+PH-3-PPP binding at 25°C is time-dependent, with a plateau in binding attained within 45 min, the incubation period employed routinely for autoradiographic studies. (+PH3-PPP binding to brain sections is saturable with a single component of binding evident by Scatchard analysis. The dissocia-

1762

Gundlach et al.

Vol. 6, No. 6, Jun. 1986

tion constant (KJ is 30 nM, similar to values obtained in brain homogenates (Largent et al., 1984). The estimated maximal number of binding sites (B,,,) is 100-l 50 fmol/l2 Mm sag&al section, consistent with levels found in brain homogenates. The drug specificity of ( +)3H-3-PPP binding in brain sections is also similar to that of homogenates. The most potent inhibitor of binding is haloperidol (IC,, = 5 nM). As in homogenates, the stereospecificity for opioids is opposite to that of conventional opiate receptors, with the (+)isomer of cyclazocine being 10 x more potent than the (-)isomer. While 3-PPP displays pharmacologic activity at dopamine receptors (see introduction), (+PH-3-PPP binding in brain sections, as well as homogenates, shows a stereospecificity opposite to that observed at dopamine receptors for the neuroleptic butaclamol, with the (-)isomer 50 x more potent than the (+)isomer.

Autoradiographiclocalizationsof (+)3H-3-PPP binding sites in guineapig CNS

Figure 2. Autoradiogramsof (+)-‘H-3-PPP binding sitesin guineapig spinal cord. Sectionsare shown at the level of upper (A) and lower (I?) cervical,thoracic (Q, andlumbar (0) spinal cord. Note the high density of ( +)3H-3-PPP-associatedsilver grainsover the largecellsin the ventral root (vr) a gray matter, particularly the ninth layer of Rexed (R9; arrowheads)and dorsalroot ganglia(drg)at all levelsof the cord. Binding is lower in the dorsal horn in layers of Rexed l-5 in upper and lower cervical, and layers l-3 in thoracic and lumbar (RI, 3, 5; asterisks) spinal cord.Further detailsaregiven in legendto Figure 1andin Results. Calibration bar. 1 mm.

Discrete localizations of (+)3H-3-PPP binding sites are observed throughout the guinea pig CNS, confirming and extending our earlier observations (Largent et al., 1984)-see Figures 1 and 2 and Table 2. In the cerebral cortex, intense binding is associated with the pyramidal cell layers, especially in the pyriform (or primary olfactory) cortex (Fig. 1, A-D). Superficial layers in the cingulate, anterior and posterior (Fig. 1, B-F), and retrosplenial (Fig. 1, G-H) cortices are also heavily labeled. In contrast to the distribution of dopamine receptors, relatively little binding is observed in the caudate-putamen (Fig. 1, B-D), although moderate levels are present in the ventral pallidum and the globus pallidus (Fig. 1D). In the hypothalamus and related areas, the medial preoptic, preoptic, supraoptic, and paraventricular nuclei are intensely labeled (Fig. lD), as are the lateral mamillary bodies (Fig. 1E). However, receptor labeling is not uniform throughout the hypothalamus, as more medially the mamillary bodies are almost devoid of autoradiographic grains. Most of the thalamus is poorly labeled, with the ventral posterior nucleus being nearly devoid of grains (Fig. 1E). By contrast, substantial binding occurs in the lateral reticular nucleus of the thalamus as well as the zona incerta of the subthalamus. The choroid plexus is also heavily labeled by ( +)3H3-PPP (Fig. 1D). Many structures associated with the limbic system are heavily labeled. For instance, the lateral septal nucleus has a high grain density, as do the medial septal area, the diagonal band, and parts of the hippocampus. Within the hippocampus, grain densities are restricted to the pyramidal cell layers (CAl-4) and the granular layer of the dentate gyrus (Fig. 1, E, F). The central gray of the midbrain is intensely labeled, and moderate binding occurs in the subiculum (Fig. 1, F-H). Within the hindbrain, intense punctate binding is seen over cranial nerve nuclei, including the hypoglossal, vestibular, facial, abducens, trochlear, and oculomotor nuclei, the motor nucleus of the trigeminal nerve, motor nucleus of the vagus, and the nucleus ambiguus (Fig. 1, G-L). Other brain stem areas with high levels of binding include the red nucleus, the pontine nuclei, the pontine and medullary reticular formation, and the superior colliculus. The cerebellum displays a discrete band of labeling associated with the Purkinje cell layer (Fig. 1, F-L). A substantial grain density overlies the granule cell layer; this contrasts with the molecular layer, which has low binding levels. Moderate grain densities occur in some of the deep cerebellar nuclei, such as the lateral cervical nucleus. In spinal cord, the highest concentrations of grains occur in the dorsal root ganglia at all levels examined (Fig. 2). In most sections the grains appear concentrated in the peripheral zone, with the central portion of the ganglion, containing fibers of passage,displaying fewer grains (seebelow). Radiographic grains


The Journal of Neuroscience Table 3. Effect of neurotoxin

lesions on the amount of (+yH-3-PPP

biiding

1763

in selected brain regions

Decreasein Structure examined

(+)‘H-3-PPP binding (%)

Pyramidal cell layer Dentate gyrus granularlayer

85 t 3** 82 k 3**

Ibotenic acid

Striatum Substantianigra

68 + 7** 8 f 2*

6-OHDA

Striatum Substantianigra

17 +- 4* 32 k 2**

Lesioned area

Toxin

Hippocampus”

Quinolinic

Striatum Striatum

acid

Pars compacta Lesions were produced in the indicated areas with the respective neurotoxins, as described in Materials and Methods, and animals were sacrificed 4 d (hippocampus) or 14 d (striatum) later. The effect of the lesion was evaluated in the ipsilateral region of interest and compared with the contralateral control region. Lesion effects were evaluated in at least four-eight brain sections Corn each of two to three animals and quantitated from the autoradiogram. Data are reported as the percentage decrease (GEM) in (+)3H3-PPP binding due to the lesion, relative to control, and are significantly different from control by at least *p < 0.005 or **p =C0.001 (one-tailed Student’s t test). n No significant change occurred in the amount of binding in the stratum radiatum.

are associated exclusively with gray matter areas throughout the spinal cord and are more dense in the ventral than the dorsal horn. Autoradiographic localizations of (+)jH-3-PPP binding sites in rat CNS As marked species differences in receptor binding sites for a variety of ligands have been described, we also examined the localization of (+)3H-3-PPP binding sites in the rat CNS (Fig. 3). Localizations are closely similar in rat and guinea pig CNS, with only quantitative differences between the two species. Very high grain densities occur over motor nuclei, such as the motor nucleus of the trigeminal nerve, and facial and hypoglossal nuclei (Fig. 3, H-K), as in the guinea pig. In the hippocampus of the rat, as in the guinea pig, grain densities are most concentrated in the pyramidal cell layer (Fig. 3, D-F). While the guinea pig has high levels of receptors in the Purkinje cell layer of the cerebellum (see below), relatively less binding is apparent in the rat cerebellum. However, the limited amount ofbinding appears primarily associated with the Purkinje and granule cell layers (Fig. 3, H-K). In the rat brain stem, pontine and reticular nuclei display lower (+)3H-3-PPP binding relative to the intense labeling of these areas in the guinea pig. The distribution of (+YH3-PPP-associated grain densities in the spinal cord of the rat is similar to that seen in the guinea pig (Fig. 3L). Cellular localization of (+)‘H-3-PPP binding sites The cellular localization of receptor binding sites was further investigated by histological examination of specific areasofguinea pig and rat brain under high-power magnification and comparison with the corresponding autoradiographic grain distributions. In all areas examined (i.e., the hippocampus, cerebellum, and brain stem), grains appear localized exclusively to neuronal cell bodies (Figs. 4 and 5). In the guinea pig cerebellum, the highest concentration of ( +)3H-3-PPP binding is over the Purkinje celI layer, with moderate to low concentrations associated with the granule cell and molecular layers, respectively (Fig. 4B). The spinal cord displays grains primarily over large motor neurons in the ventral horn. In dorsal root ganglia, (+)3H-3PPP binding is associated with the sensory cells and not with the white matter tracts passing through the ganglion (Fig. 40). The binding is so intense that, even with coverslips, it is difficult to differentiate between the sensory and satellite cells. Given the association of ( +)sH-3-PPP binding with gray matter regions elsewhere in the brain, binding is likely to be to the sensory cells (seebelow). (+)3H-3-PPP binding in all the cranial nerve nuclei

selectively overlies large motor cells, for example, the hypoglossal (Fig. 5A), ambiguus (Fig. 5B), and facial (Fig. 5D) nuclei. Radiographic grains in the hippocampus are selectively associated with pyramidal cells and the granule cells of the dentate gyrus (Fig. 6), while, in the pars compacta of the substantia nigra, receptors appear localized to dopamine neuron cell bodies (see below). Eflects of neurotoxin lesions on (+)SH-3-PPP binding sites in rat brain The selective association of autoradiographic grains with neuronal cell bodies in many brain regions suggeststhat receptors are primarily localized to neuronal cells. However, grains could be localized also to nerve terminals synapsing on neuronal soma. Accordingly, selective lesions of intrinsic neurons were employed to investigate this possibility. In the hippocampus, quinolinic acid lesions abolish autoradiographic grains over the pyramidal cell layer of the hippocampus and the granule cell layer of the dentate gyrus (Fig. 6, Table 3). The loss of binding in hippocampal pyramidal cell layers with quinolinic acid treatment strongly suggests that receptors are selectively associated with the pyramidal cells. Furthermore, lesions of the entorhinal cortex and lateral septum, both sources of major neuronal projections to the hippocampus, produce no change in grain patterns in the hippocampus. Thus, within the hippocampus, grains appear to be primarily, if not exclusively, associated with pyramidal cells. In a single animal, kainic acid injected into the cerebellum depleted receptor binding associated with the granule and Purkinje cell layers by about 50%, with no effect on the low levels of binding to the molecular layer (data not shown). To evaluate ( +)3H-3-PPP-associated grain localizations in the striatonigral system, either ibotenic acid or 6-hydroxydopamine was administered into the corpus striatum to lesion intrinsic neurons or dopaminergic tierents, respectively. Both the corpus striatum and substantia nigra were examined in each lesioned animal for changes in (+)jH-3-PPP binding (Table 3). Ibotenic acid administered into the corpus striatum substantially depleted striatal autoradiographic grains with a much smaller effect on binding in the substantia nigra, suggesting an association of striatal (+)3H-3-PPP binding primarily with intrinsic neurons that do not project to the substantia nigra. Striatal injection of 6-hydroxydopamine, which destroys dopamine nerve terminals in the corpus striatum and produces a retrograde degeneration of dopamine containing cells in the pars compacta of the substantia nigra, significantly depleted ( +)3H3-PPP binding in the ipsilateral pars compacta of the substantia

1764

Gundlach et al.

Vol. 6, No. 6, Jun. 1986

Figure 3. Autoradiograms of (+)‘H-3-PPP binding in rat brain and spinal cord. Representative sections are presented in a rostrakaudal progression for brain (A-K) and for cervical and thoracic spinal cord (L). Nonspecific binding of (+)‘H-3-PPP in the presence of 1 PM haloperidol is shown at a similar exposure (M). Abbreviations are as in legend to Figure 1 and as follows: AHy. anterior hypothalamic area; Am, amygdala; Arc, arcuate n.; BL, basolateral amygdaloid n.; Dh, dorsal horn, spinal cord; Hi, hippocampus; LC, locus coeruleus; LHy, lateral hypothalamic area; PV, paraventricular thalamic n.; RM, raphe magnus n.; R9, ninth layer of Rexed, spinal cord. Further details are given in the legend to Figure 1 and in the text. Calibration bar, 2 mm.


Figure 3.


1765

Continued.

nigra, with a relatively smaller loss of binding evident in the corpus striatum (Table 3). In the same animals, striatal 3Hmazindol binding, associated with dopamine uptake sites on dopaminergic nerve terminals (Javitch et al., 1985), is abolished, demonstrating the effectiveness of the lesion. This suggeststhat (+)3H-3-PPP binding is present on the cell bodies of dopamine containing neurons in the pars compacta of the substantia nigra and that a small portion of ( +)3H-3-PPP binding (- 20%) in the corpus striatum is present on dopaminergic terminals. The possible association of sigma receptors with other biogenie amine systems was also investigated. Rats were treated with p-chloroamphetamine, which destroys serotonin neurons (Kohler et al., 1978; Neckers et al., 1976) and N-[2-chloroethyllN-ethyl-2-bromobenzylamine (DSP-4), which selectively destroys norepinephrine-containing neurons (Hallman et al., 1984; Jonsson et al., 1981), and (+)3H-3-PPP binding was measured in whole-brain homogenates. Neither of these treatments markedly affected binding levels, despite their depletion of serotonin and norepinephrine concentrations, respectively, indicating significant lesions of these neuronal systems (data not shown). Discussion Binding sites for (+)3H-3-PPP in both guinea pig and rat CNS display very discrete localizations. While it is difficult to generalize about the type of structures labeled, the highest densities of radiographic grains appear to be associated with structures of the limbic system, cranial nerve motor and sensory nuclei, as well as reticular nuclei at all levels of the brain stem. Furthermore, the sensory dorsal root ganglia and the motor cells in the ventral horn of the spinal cord are heavily labeled. Highpower magnification studies of binding patterns indicate a selective localization of grains over neuronal cell bodies, a finding confirmed by a variety of lesion studies. Grain densities are highest in nuclei with large neuronal cells, such as the supraoptic

and paraventricular nuclei of the hypothalamus, the pyramidal cells of the hippocampus and cortex, and Purkinje cells of the cerebellum. Previous studies (Gundlach et al., 1985; Largent et al., 1984, in press) revealed that the drug specificity of ( +)3H-3-PPP binding sites is very similar to sigma receptor binding sites identified with (?) and (+)3H-SKF 10,047, 3H-haloperidol, and 3Hethylketocyclazocine (Gundlach et al., 1985; Largent et al., 1984, in press; Su, 198 1, 1982; Tam, 1985; Tam and Cook, 1984). Other studies have suggestedthat psychotomimetic opioids, such as (+)3H-SKF 10,047, label binding sites resembling PCP receptors (Itzhak et al., 1985; Mendelsohn et al., 1985; Zukin et al., 1984) and differ from sigma sites. This discrepancy appears resolved by recent findings that (+)3H-SKF 10,047 labels two binding-site populations (Gundlach et al., 1985; Largent et al., in press). The high-affinity sites have the pharmacological specificity of sigma binding sites as defined by their labeling with 3H-haloperidol and (+)‘H-3-PPP, while the low-affinity sites represent PCP receptors. Thus, the binding sites examined in the present study are those defined as non-PCP sigma receptor binding sites. Studies of the effects of 3-PPP in rodents suggestthat the drug interacts with autoreceptors on dopamine neurons (Amt et al., 1983; Hjorth et al., 1981, 1983, 1985). In the substantia nigra, sigma receptor binding sites appear localized to the dopaminecontaining cells of the pars compacta. Conceivably, the localization of sigma receptors in the nigra, as well as the cerebellum and other relevant regions, might relate to the effects of 3-PPP on locomotion. However, recent studies indicate that dopaminergic effects of 3-PPP are blocked by (+)butaclamol but not by the (-)isomer (Hjorth et al., 1985), in contrast to the stereospecificity. observed for butaclamol isomers at sigma receptors. Whether the sigma binding sites are responsible for mediating

Figure 4. Localization of (+)‘H-fPPP binding sites in guinea pig cerebellum and dorsal root ganglion. A, Cerebellum tissue stained with (Gr) and molecular (MO) layers, and also white matter (wm). The Purkinje cell layer (P/c) is located between the borders of these two layers corresponding autoradiogram shows the intense binding of (+)“H-3-PPP to the Purkinje cell layer and the differential binding to the granule bar, 150 pm. C, Photomicrograph of dorsal root ganglion reveals large, pale sensory cells (Se; arrowheads) surrounded by the small, dark (wm) passing through the ganglion. 0, The autoradiogram reveals intense binding of (+)‘H-3-PPP over the sensory/satellite cells relative Calibration bar, 150 pm.

toluidine blue shows the granule cell (urrowhends). B, Photomicrogmph of cell and molecular layers. Calibration satellite cells and white matter tracts to the low levels over white matter.

Figure 5. Localization of (+PH-3-PPP binding sites in rat hypoglossal, ambiguus, and facial nuclei. Light-field photomicrographs of the hypoglossal (A) and ambiguus (B) nuclei at low magnification show large numbers of silver grains selectively located over large cells in these regions. Calibration bar, 120 pm. Higher-magnification photomicrographs of individual cells in the facial nucleus reveal the characteristic pale-stained motor neurons (C) and the concentration of silver grains over the non-nuclei regions of the cells (0). Calibration bar, 40 Wm.

of (A) control and (c) lesioned (200 Figure 6. Effect of a quinohnic acid lesion on the binding of (+PH-3-PPP in the rat hippocampus. Photomicrographs nmol, 4 d) sides of the hippocampal formation (stained with toluidine blue) demonstrate the extensive loss of pyramidal (Py) and granule (GrDG) cells caused by the lesion. Autoradiograms show the distribution pattern of (+))H-fPPP binding sites for both the control (B) and injected (0) sides of the hippocampus and reveal the elimination of binding coincident with the loss of hippocampal pyramidal cells and granule cells of the dentrate gyms. Calibration bar, 0.5 mm.

G 3

$

-9

$ “0, 5



the sigma-like psychotomimetic effects of opioids is difficult to establish. Human data are limited, and it is unclear whether the behavioral effects of drugs in animals faithfully mimic those in humans. However, the sigma binding sites do show stereoselectivity for opioids, with the (+)isomer being more potent; this corresponds to the greater potency of the (+)isomers of opioids in producing putative sigma-behavioral effects (Brady et al., 1982; Katz et al., 1985; Slifer and Balster, 1983). Recently, on the basis of autoradiographic results revealing sigma receptor binding sites on cerebellar Purkinje cells, the in vivo function of sigma receptors was investigated. Iontophoresis of (+)3-PPP onto cerebellar Purkinje cells decreasestheir spontaneous firing rate, an effect potently blocked by haloperidol and (+)SKF 10,047, but only weakly by (-)SKF 10,047 (Lee et al., 1985). Moreover, the (+)isomer of 3-PPP is more potent than the (-)isomer, similar to their relative potencies in vitro at sigma receptor binding sites. This in vivo effect of (+)3-PPP corresponds with the in vitro autoradiographic localization of sigma sites to cerebellar Purkinje cells and the pharmacological profile of sigma sites, thus suggesting that these in vivo inhibitory effects of (+)3-PPP are mediated through sigma receptors.

References Arnt, J., K. P. Bogeso, A. V. Christensen, J. Hyttel, J. J. Larsen, and 0. Svendsen (1983) Dopamine receptor agonistic and antagonistic effects of 3-PPP enantiomers. Psychopharmacology 81: 199-207. Brady, K. T., R. L. Balster, and E. L. May (1982) Discriminative stimulus properties of stereoisomers of N-allylnormetazocine in phencyclidineltrained squirrel monkeys and rats. Science 215: 178-l 80. Gearv. W. A.. and G. F. Wooten (1983) Quantitative film autoradi&aphy of opiate agonist and antagonist binding in rat brain. J. Pha&acbl. Exp. Ther: 225: 234-240.Gundlach, A. L., B. L. Largent, and S. H. Snyder (1985) Phencyclidine and sigma opiate receptors in brain: Biochemical and autoradiographical di%‘erentiation. Eur. J. Pharmacol. 113: 465-466. Haertzen, C. A. (1970) Subjective effects of narcotic antagonists cyclazocine and nalorphine on the addiction research center inventory (ARCI). Psychopharmacologia 81: 366-377. Hallman, H., E. Sundstrom, and G. Jonsson (1984) Effects of the noradrenaline neurotoxin DSP 4 on monoamine neurons and their transmitter turnover in rat CNS. J. Neural Transm. 60: 89-102. Haubrich, D. R., and A. B. Pflueger (1981) The autoreceptor control of dopamine synthesis. Mol. Pharmacol. 21: 114-120. Hjorth,-S., A. Carlsson, H. Wikstrom, P. Lindberg, D. Sanchez, U. Hacksell. L. E. Arvidsson. U. Svensson. and J. L. G. Nilsson (198 1) 3-PPP, a new centrally acting dopamink receptor agonist with‘selec: tivity for autoreceptors. LifeSci. 28: 1225-1238. Hiorth. S.. A. Carlsson. H. Wikstrom. P. Lindbera. D. Sanchez, U. “Hacksell, L. E. Arvidsson, U. Svenson, and J. L. G. Nilsson (1983) Central dopamine receptor agonist and antagonist actions of the enantiomers of 3-PPP. Psychopharmacology 81: 89-99. Hjorth, S., D. Clark, and A. Carlsson (1985) Lack of functional evidence for the involvement of sigma opiate receptors in the actions of the 3-PPP enantiomers on central dopaminergic systems: Discrepancies between in vitro and in vivo observations. Life Sci. 37: 673684. Itzhak, Y., J. M. Hiller, and E. J. Simon (1985) Characterization of specific binding sites for [‘H](d)-N-allylnormetazocine in rat brain membranes. Mol. Pharmacol. 27: 46-52. Iwamoto, E. T. (198 1) Locomotor activity and antinociception after putative mu, kappa and sigma opioid receptor agonists in the rat: Influence of dopaminergic agonists and antagonists. J. Pharmacol. Exp. Ther. 217: 45 l-460. Javitch, J. A., S. M. Strittmatter, and S. H. Snyder (1985) Differential visualization of dopamine and norepinephrine uptake sites in rat brain using [3H]mazindol autoradiography. J. Neurosci. 5: 15 13-l 521. Jonsson. G.. H. Hallman. F. Ponzio. and S. B. Ross (1981) DSP4 (N[2-chloroethyl]-N-ethyl-2-bromobenzylamine)-a useful henervation tool for central and peripheral noradrenaline neurons. Eur. J. Pharmacol. 72: 173-188. Katz, J. L., R. D. Spealman, and R. D. Clark (1985) Stereoselective

1769

behavioral effects of N-allylnormetazocine in pigeons and squirrel monkeys. J. Pharmol. Exp. Ther. 232: 452-461. Keats, A. S., and J. Telford (1964) Narcotic antagonists as analgesics. Clinical aspects. In Molecular Modi$cation in Drug Design. Advances in Chemistry, Ser. 45, R. F. Gould, ed., pp. 170-l 76, American Chemical Society, Washington, DC. Kohler, C., S. B. Ross, B. Srebro, and S.-O. Ogren (1978) Long-term biochemical and behavioural effects of p-chloroamphetamine in the rat. Ann. NY Acad. Sci. 305: 645-663. Kuhar, M. J., P. J. Whitehouse, J. R. Unnerstall, and H. Loats (1984) Receptor autoradiography: Analysis using a PC-based imaging system. Sot. Neurosci. Abstr. IO: 558. Largent, B. L., A. L. Gundlach, and S. H. Snyder (1984) Psychotomimetic opiate receptors labeled and visualized with (+)[‘H]3-(3Hydroxyphenyl)-N-( 1-propyl)piperidine. Proc. Natl. Acad. Sci. USA 81: 4983-4987. Largent, B. L., A. L. Gundlach, and S. H. Snyder (in press) Pharmacological and autoradiographic discrimination of sigma and phencyclidine receptor binding sites in brain with (+)[3H]SKF10,047,(+)~H]3-PPP and [,H]TCP. J. Pharmacol. Exp. Ther. Lee, M., A. L. Gundlach, and J. C. Strahlendorf (1985) Electrophysiological effects of 3-(3-hydroxyphenyl)-N-( 1-propyl)piperidine on spontaneously active cerebellar Purkinje neurons. Sot. Neurosci. Abstr. 11: 690. Luparello, T. J. (1967) Stereotaxic Atlas of the Forebrain of the Guinea Pig, Karger, Basel. Markstein, R., and D. Lahaye (1983) In vitro effect of the racemic mixture and the (-)enantiomer of N-n-propyl-3-(3-hydroxyphenyl)piperidine (3-PPP) on postsynaptic dopamine receptors and on a presynaptic dopamine autoreceptor. J. Neural Transm. 58: 43-53. Martin, B. R., J. S. Katzen, J. A. Woods, H. L. Tripathi, L. S. Harris, and E. L. May (1984) Stereoisomers of [3H]-N-allylnormetazocine bind to different sites in mouse brain. J. Pharmacol. Exp. Ther. 231: 539-544. Martin, W. R., C. G. Eades, J. A. Thompson, R. E. Huppler, and P. E. Gilbert (1976) The effects of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog. J. Pharmacol. Exp. Ther. 197: 5 17-532. McPherson, G. A. (1983) A practical computer-based approached to the analysis of radioligand binding experiments. Comput. Prog. Biomed. 17: 107-114. Mendelsohn, L. G., V. Kalra, B. G. Johnson, and G. A. Kerchner (1985) Sigma opioid receptor: Characterization and co-identity with the phencyclidine receptor. J. Pharmacol. Exp. Ther. 233: 597-602. Mulder, A. H., R. Draper, P. Sminia, A. N. M. Schoffelmeer, and J. C. Stoof (1985) Agonist and antagonist effects of 3-PPP enantiomers on functional dopamine autoreceptors and postsynaptic dopamine receptors in vitro. Eur. J. Pharmacol. 107: 291-297. Neckers, L. H., L. Bertilsson, S. H. Koslow, and J. L. Meek (1976) Reduction of tryptophan hydroxylase activity and 5-hydroxytryptamine concentration in certain rat brain nuclei after P-chloroamphetamine. J. Pharmacol. Exp. Ther. 196: 333-338. Paxinos, G., and C. Watson (1982) The Rat Brain in Stereotaxic Coordinates, Academic, Sydney. Ralis, H. M., R. A. Beesley, and Z. A. Ralis (1973) Techniques in Neurohistology, Butterworth’s, London. Schwartz, R., T. H&felt, K. Fuxe, G. Jonsson, M. Goldstein, and L. Terenius (1979) Ibotenic acid-induced neuronal degeneration: A morphological and neurochemical study. Exp. Brain Res. 37: 199216. Schwartz, R., W. D. Whetsell, Jr., and R. M. Mangano (1983) Quinolinic acid: An endogenous metabolite that produces axon-sparing lesions in rat brain. Science 219: 3 16-3 18. Shannon, H. E. (1983) Pharmacological evaluation of N-allylnonnetazocine (SKF 10,047) on the basis of its discriminative stimulus properties in the rat. J. Pharmacol. Exp. Ther. 225: 144152. Slifer, B. L., and R. L. Balster (1983) Reinforcing properties of stereoisomers of the putative sigma agonists N-allylnomretazocine and cyclazocine in rhesus monkeys. J. Pharmacol. Exp. Ther. 225: 522-528. Sminia, P., and A. H. Mulder (1983) Failure of 3-PPP to activate dopamine autoreceptors in vitro. Eur. J. Pharmacol. 89: 183-184. Su, T.-P. (198 1) Psychotomimetic opioid binding: Specific binding of [3H]SKF-10047 to etorphine-inaccessible sites in guinea-pig brain. Eur. J. Pharmacol. 75: 81-82. Su, T.-P. (1982) Evidence for sigma opioid receptor: Binding of [‘H]SKF

1770

Gundlach et al.

10,047 to etorphine-inaccessible sites in guinea-pig brain. J. Pharmacol. Exp. Ther. 223: 284-290. Tam, S. W. (1983) Naloxone-inaccessible G receptor in rat central nervous system. Proc. Natl. Acad. Sci. USA 80: 6703-6707. Tam, S. W. (1985) (+)-[HISKF 10,047, (+)-[3Hjethylketocyclazocine, p, r, 6 and phencyclidine binding sites in guinea pig brain membranes. Eur. J. Pharmacol. 109: 33-4 1. Tam, S. W., and L. Cook (1984) (r opiates and certain antipsychotic drues mutuallv inhibit (+)-PHlSKF 10.047 and PHlhaloueridol bindingIn guinea pig brain mimdranes. Proc. Natl: Acad. Sci. USA 81: 5618-5621. Ungerstedt, U. (1971) Postsynaptic supersensitivity after 6-hydroxydopamine induced deaeneration of the nigro-sttiatal dopamine system. Acta Physiol. S&d. (Suppl.) 367: 69-73. Unnerstall, J. R., D. L. NiehofT, M. J. Kuhar, and J. M. Palacios (1982) Quantitative receptor autoradiography using [3H]Ultrofilm: Appli-

Vol. 6, No. 6. Jun. 1986

cation to multiple benzodiazepine receptors. J. Neurosci. Methods 6: 59-73. Vaupel, D. B. (1983) Naltrexone fails to antagonize the effects of PCP and SKP 10,047 in the dog. Eur. J. Pharmacol. 92: 269-274. Young, G. A., and N. Kbaxan (1984) Differential neuropharmacological effects of mu, kappa and sigma opioid agonists on cortical EEG power spectra in the rat. Neuropharmacology 23: 116 l-l 165. Zukin, S. R., and R. S. Zukin (1979) Specific [‘Hlphencyclidine binding in rat central nervous system. Proc. Natl. Acad. Sci. USA 76: 53725376. Zukin, R. S., and S. R. Zukin (198 1) Demonstration of [3H]cyclazocine binding to multiple opiate receptor sites. Mol. Pharmacol. 20: 246254. Zukin, S. R., K. T. Brady, B. L. Slifer, and R. L. Balster (1984) Behavioral and biochemical stereoselectivity of sigma opiate/PCP receptors. Brain Res. 294: 174-177.